An essential function of the intestinal mucosa is to regulate Na absorption appropriately in response to potentially large variations in daily oral intake and metabolic perturbations. In this competitive renewal application, we will examine the cellular events involved in the regulation of the apical membrane Na-H exchangers, NHE2 and NHE3, in this process, as they are major pathways for non-nutrient dependent Na absorption. We hypothesize that their acute regulation is in large part dependent on an orchestrated movement in and out of the apical membrane that is highly dependent on actin cytoskeleton and other integral membrane and adaptor proteins. We also hypothesize that they are not randomly distributed in the apical membrane, but found in highly ordered lipid domains (non-caveolin lipid rafts) and clathrin-coated regions. Their distribution in these specialized membrane domains is essential for their function and regulation, specifically in clustering them with other transporters such as the anion exchanger, DRA (Down-Regulated in Adenoma), the dipeptidy1 transporter hPepT1, adaptor proteins (E3KARP and NHERF), and other key molecules such as v- and t-SNARES required for membrane endo- and exocytosis. Finally, we believe disease-related perturbations of intestinal Na absorption are mediated by their specific effects on components of these events and/or proteins. To examine these hypotheses, we propose three specific aims: (1) To define the process and mechanisms of induced membrane trafficking of NHE2 and NHE3 involved in their regulation by intestinal epithelial cells, (2) To examine the potential functional and physical interactions between NHEs and DRA and hPepT1, two transporters brush border transporters that we believe are coupled with and regulated by apical NHEs, and (3) To define mechanisms of disease-related perturbations in NHE function and regulation using two experimental conditions - inflammation-associated oxidant stress and metabolic acidosis, which inhibit and enhance gut Na absorption, respectively. We will use a combination of functional, biochemical, and imaging approaches to provide detailed mechanistic and novel insights into how NHEs are regulated physiologically, their relative roles in coupled dipeptide and anion transport, how their dysfunction and aberrant regulation by certain disease processes ultimately cause alterations in net water and electrolyte transport by the gut.